Fig. 2.

rMRVs with in-frame 5′ or 3′ ORF region duplication, with or without in-frame 2ALE insertion. (A) Schematics of the modified M1 or S3 plus-strand RNAs used for creating these rMRVs. See Fig. 1 for labeling and color schemes. The in-frame outer repeat, inner repeat, and 2ALE lengths (in nt) are now also labeled in each schematic. (B) Electropherogram of genomic dsRNAs from native MRV-T3D (purified virions) and rMRV M1/3′153. The band corresponding to the slower-migrating, lengthened M1 segment of M1/3′153 is indicated (arrowhead). (C) Parallel growth in L929 cells by rMRVs WT (white bars) and M1/3′153/2A (black bars). (D) Electropherogram of genomic dsRNAs from rMRVs WT and S3/2A/5′174. The band corresponding to the more slower-migrating, lengthened S3 segment of S3/2A/5′174 is indicated (arrowhead). (E) Fluorogram of [³⁵S]Met-labeled in vitro translation products of plus-strand RNAs transcribed from the indicated plasmid constructs of MRV segments. Constructs pS3/2A/5′174 and pS3/2AΔ1/5′174 are identical except for a 1-nt deletion in the latter, which inactivates the 2ALE and places the downstream σNS ORF out of frame with the 2ALE and upstream sequences. The lack of a full-length σNS band translated from pS3/2AΔ1/5′174 RNA is indicated (gray arrowhead).